1. Field of the Invention
The present invention relates to gram-positive bacterial strains resistant to 5-fluorodeoxyuridine, and to methods of producing such bacterial strains.
2. Description of the Related Art
Commensal Gram-positive bacteria are currently being developed as live vaccine vectors able to colonize mucosal surfaces and stimulate a secretory IgA, as well as a systemic immune, response to a recombinant antigen displayed on the surface of these organisms. One such promising live vaccine delivery system candidate is the oral bacterium Streptococcus gordonii, a component of human dental plaque which is able to colonize most oral sites. One important issue related to the utilization of these organisms as live vaccine vectors is the need to develop non-antibiotic selectable markers to employ during the manipulation of these recombinant organisms in the research and clinical laboratories, as well as to monitor colonization in animal studies and possible dispersal of these organisms in the environment.
Thymidine kinase (TK; EC 2.7.1.21) is a key enzyme in the salvage pathway of pyrimidine nucleotide metabolism which catalyzes the formation of thymidylate from thymidine and ATP. Previous studies in both Escherichia coli and Salmonella typhimurium have suggested it is possible to select for tdk mutations in essentially any genetic background on the basis of their resistance to the pyrimidine analog fluorodeoxyuridine. Presumably, when FUDR is phosphorylated by TK to 5-fluorodeoxyuridine monophosphate it inhibits thymidylate synthetase, the final enzyme in the de novo synthetic pathway to thymidylate synthesis. The resultant absence of a source of preformed thymidine will deplete the cell of the essential metabolite TMP and thus, the selection pressure for the mutation of tdk. Indeed, previous isolates of Escherichia coli, resistant to FUDR, all possessed mutations that mapped to a single locus at 27.5 minutes on the E. coli genetic map and were deficient in thymidine kinase activity. This suggests that the usual mechanism for FUdR-resistance is the loss of thymidine kinase activity and that all mutations in FUdR.sup.r mutants of E. coli occur at the tdk locus.
Since the gene encoding thymidine kinase of S. gordonii has been cloned and the nucleotide sequence determined, we sought to determine if the selection for FUdR-resistant strains of S. gordonii was possible and, if so, to evaluate it as a dominant selectable marker.